The potential of crop residue fertilization as a carbon sequestration technology was evaluated in a central Ohio Luvisol (fine, mixed, mesic Aeric Ochraqualf). Wheat (Triticum aestivum L.) straw was applied at three rates (bare: 0; low: 8 Mg; and high: 16 Mg ha−1yr−1) without (−F) and with (+F) addition of 244 kg N ha−1yr−1. The treatments were triplicated in a completely randomized block design. We report on the impact of these treatments on the total stocks and biochemical attributes of soil organic C (SOC) in the 0 to 10-cm soil layer after 4 years. Microbial biomass C (MBC) and mineralizable C (MinC), as measures of the labile C pools, were used as biochemical indices. SOC stocks, MBC and MinC were generally lowest in the bare plots, and more so with fertilization of these treatment plots. The addition of wheat straw and fertilizer had a positive and interacting effect on SOC stocks. With low mulch application, SOC stocks were similar (25.6 Mg C ha−1) in the −F and +F plots. However, when the mulch rate was high, further SOC accretion (28.2 Mg ha−1) occurred only in the +F plots. Residue-C conversion into SOC averaged 14% and 32% without and with fertilization, respectively. Positive effects of wheat straw application on MBC and MinC110 dayswere also found. Although not significant, MBC was generally lower in the fertilized compared with unfertilized treatments. Fertilization of wheat residue resulted in a 1.5-fold increase in MinC, and a 1.3 to 4 times increase in the metabolic quotient (unit of MinC per unit of MBC per day), suggesting alteration in the soil microbial community composition and less efficient C processing with fertilization. The temporal variation of these soil biochemical attributes and the fate of the labile C compounds that evolved with fertilization of residue will determine the long-term sustainability of residue fertilization practice on C sequestration in soils.